I Psc
- 2. Introduction Stem Cells Ability to self renew, to make exact copies of itself through cell division. Ability to develop into different types of mature cells. Image adopted from:- http://clearlyexplained.com/nature/life/cells/stemcelltree.gif
- 3. Stem Cells Have the ability to specialize in all postembryonic tissues and organs, and also extra embryonic tissues. E.g.- zygote Capable of giving rise to most tissues of an organism. E.g.- ES cells Multipotent Stem cells that can differentiate into many family of Can only differentiate into single type of cell cells, E.g.– Hematopoietic stem cells but have the property of self-renewal, E.g.- mucus membrane cells and skin stem cell Adopted from:- http://www.wikipedia.org
- 5. Three methods of inducing pluripotency in somatic cells. ©2008 by The Royal Society Adopted from:- Yamanaka S Phil. Trans. R. Soc. B 2008;363:2079-2087
- 6. Induced pluripotent stem cells (iPSCs) are typically adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.
- 7. iPSc-made from mouse-> lead to the creation of beating cardiomyocyte, skin and almost every kind of cell you can imagine. iPSc made from human-> also lead to the creation of various cell types in body, in addition even produced gamete! iPSc created live organism-> “ Xiao Xiao / Tiny”
- 8. Adopted from: Cell 126, 663–676, August 25, 2006 ©2006 Elsevier Inc. 663
- 9. 24 key factors are responsible for pluripotency Tcl 1 ecat 1 Dnmt Ecat 15 3L Ecat 8 Nanog Gdf 3 Ecat β 15-2 Rex 1 catenin fth 17 Grb 2 lif Sall 4 Sox 15 Fbx 15 Utf 1 esg 1 sox 2 Stat 3 c-myc oct3/4 ERas klf 4
- 11. Fbx 15 locus Fbx 15 locus β-geo β-geo mEF/mAF ES- like cell Retroviral infection & G418 selection 5’LTR cDNA 3’LTR
- 12. Dnmt ecat 1 Ecat 15 Nanog Tcl 1 Ecat 8 fth 17 3L Sall 4 β catenin Fbx 15 locus Fbx 15 locus Grb 2 β-geo β-geo Rex 1 Gdf 3 mEF/mAF ES- like cell Retroviral Ecat 15-2 infection & esg 1 G418 selection Utf 1 lif 5’LTR cDNA 3’LTR Fbx 15 Sox 15 Klf 4 C-myc Oct3/4 Sox 2 ERas Stat 3
- 14. Fbx 15 locus Fbx 15 locus β-geo β-geo mEF/mAF iPSC Retroviral cocktail infection & G418 selection 5’LTR cDNA 3’LTR Tcl 1 Fbx 15 Utf 1 Rex 1 Dnmt Nanog 3L β Ecat 8 esg 1 Ecat 15-2 Stat 3 Grb 2 catenin fth 17 Ecat 15 lif C-myc Oct3/4 ecat 1 Klf 4 Sox 2 ERas Gdf 3 Sox 15 Sall 4
- 16. NOW THEY TRIED TO SEE THE MINIMUM NUMBER OF FACTORS THAT COULD INDUCE PLURIPOTENCY
- 17. Fbx 15 locus Fbx 15 locus β-geo β-geo mEF/mAF iPSC Retroviral cocktail infection & G418 selection 5’LTR cDNA 3’LTR Klf 4 C-myc Oct3/4 Sox 2
- 18. Results Multiple number of G418-resistant colonies were observed after transduction with a combination of these four factors (oct3/4, sox2, klf4, c-myc) Cell 126, 663-676, August 25, 2006 ©2006 Elsevier Inc.
- 19. VARIOUS TISSUES PRESENT IN TERATOMAS DERIVED FROM iPSC IMMUNOSTAINING CONFIRMING DIFFERENTIATION INTO NEURAL TISSUE (NEURON & GLIAL CELLS) AND MUSCLES IN TERATOMAS DERIVED FROM iPSC IN VITRO EMBRYOID BODY FORMATION Cell 126, 663-676, August 25, 2006 ©2006 Elsevier Inc.
- 20. RT- PCR ANALYSIS OF ESC MARKER GENES IN IPSC BISULFITE GENOME SEQUENCING OF THE PROMOTER REGIONS OF GENES FROM IPSC Cell 126, 663-676, August 25, 2006 ©2006 Elsevier Inc.
- 22. In June 2007 Yamanaka et al, Kyoto University & two other independent groups Harvard, MIT & UC, Los Angeles showed successful reprogramming and production of viable chimeras Instead of Fbx15 they used Nanog as a marker of pluripotency detection DNA methylation patterns were identical to ESCs
- 23. iPSc-made from mouse-> lead to the creation of beating cardiomyocyte, skin and almost every kind of cell you can imagine. iPSc made from human-> also lead to the creation of various cell types in body, in addition even produced gamete! iPSc created live organism-> “ Xiao Xiao / Tiny”!
- 24. For humans the same procedure was followed, excepting the addition and substitution of a few other factors like:- OCT 4, SOX 2, NANOG, LIN 28 genes & lentiviral system- by Thompson J. POU5F1(OCT4), C-MYC, KLF4, SOX2 genes & retroviruses- by Yamanaka S. PCM Reprogramming factors FM Fibroblasts iPSC (NANOG-GFP) Adopted from:- http://www.systembio.com/downloads/StemCellposter.pdf
- 26. iPSc-made from mouse-> lead to the creation of beating cardiomyocyte, skin and almost every kind of cell you can imagine. iPSc made from human-> also lead to the creation of various cell types in body, in addition even produced gamete! iPSc created live organism-> “ Xiao Xiao / Tiny” !
- 27. ADOPTED FROM: Stem cells: The magic brew, Janet Rossant. Nature 448, 260-262 (19 July 2007); doi:10.1038/448260a
- 28. TETRAPLOID COMPLEMENTATION FORMS ONLY PLACENTA AND 2N EXTRA-EMBRYONIC MEMBRANE FUSION 4N 2N WHITE COAT COLOUR “XIAO XIAO “/ “ TINY “ / AN ALL - iPSC MOUSE SKIN REPROGRAMMING iPSC BLACK COAT CAN FORM ALL POTENTIAL COLOUR BLACK COAT TYPES OF CELLS IN BODY COLOUR
- 29. Defined factors Motor neurons Adopted from:- http://www.rndsystems.com/cb_detail_objectname_cb09i2_induced_pluripotent_stem_cells.aspx
- 31. Morphology- cell have round shape, large nucleolus and scant cytoplasm; colony morphology is also similar. Growth properties- iPSCs are mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs. Pluripotent Stem cell markers:- iPSCs express cell surface antigenic markers expressed on ESCs. Pluripotent Stem Cell Genes- Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT. Telomerase activity- express high telomerase activity.
- 32. Neural differentiation- βIII-tubulin, GFAP expressed (found specifically in neurons and glial cells). Cardiac differentiation- Differentiated into cardiomyocytes that spontaneously began beating. Teratoma formation- iPSCs injected into immunodeficient mice spontaneously formed teratomas after nine weeks. Embryoid body- iPSCs also form embryoid bodies and have peripheral differentiated cells. Chimeric mice- Mice with iPSC derivatives incorporated all across their bodies with 10%-90% chimerism. Tetraploid complementation- Whole, non-chimeric, fertile mice produced, although with low success rate.
- 33. Promoter demethylation- Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in iPSCs, demonstrating their promoter activity. Histone demethylation- H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated.
- 34. Adopted from:- cell 137, April 3, 2009, 13 ©2009 Elsevier Inc.
- 36. HbS /HbS HbS /HbS HbA /HbS HbS /HbS Scientists of MIT, have used iPS cells to cure sickle cell anemia. Adopted from:- 21 DECEMBER 2007 VOL 318 SCIENCE www.sciencemag.org
- 37. Whitehead Institute researchers rescued Parkinson's phenotype in rat, transplanting committed neurons free of contaminating undifferentiated cell population & thus minimizing risk of tumor formation. Reprogramming Redifferentiation Transplantation into adult brain of parkinson’s disease rat model,after removal of contaminating pluripotent cells from committed neurons using FACS
- 39. Whitehead Institute researchers produced Parkinson’s disease patient-specific stem cells free of harmful reprogramming transgenes Skin cells of parkinson’s patient Retroviruses packaged with reprogramming genes flanked by lox-p sequence iPS cells with integrated transgenes Retroviruses packaged with cre enzyme iPS cells without transgenes Differentiatng factors Dopaminergic neurons Stain Green-> class III beta-tubulin (neuron specific) Red-> tyrosine hydroxylase (dopaminergic neuron specific) Adopted from:- http://www.wi.mit.edu/news/archives/2009/rj_0305.html
- 40. Suneet Agarwal & George Daley from Children's Hospital Boston and the Harvard Stem Cell Institute were studying Inherited premature aging disorder- Dyskeratosis congenita A progeroid disease Bone marrow failure Cells lose telomerase activity due to reduced expression of TERC(Telomerase RNA Component) BUT iPSC from skin of such patients shows TERC expression three times more than diseased cell Thus simply turning the diseased skin cells into iPS cells helped restore their damaged telomeres at normal level Published online in NATURE 17/2/2010
- 41. iPS Cells can be used for toxicological studies that could save lives & money as these iPSC derived tissues can be exposed to a vast library of chemical compounds to test them as potential drugs. Human disease can be studied now in human tissues i.e. worth studying in mouse models increasing the level of accuracy. Making iPSC showed the link between stem cells and cancer cells, as the reprogramming factors are in general cancer critical genes (c-myc, klf4 and others).
- 42. No transplant rejection No ethical issues involved as no usage of embryos As a method of cloning, the nonself cytoplasmic inheritance is absent here unlike SCNT
- 43. Oncogenic effect of retroviral integration Due to c-myc 20% chimeric mice developed cancer Forced reprogramming related safety issues Teratoma related safety issues Re-differentiation related safety issue
- 45. Trans-differentiation is the process when a differentiated cell creates cells outside its established differentiation path i.e. the cell fate switches, including the interconversion of stem cells Trans-differentiation was shown earlier by converting pancreatic exocrine cell to beta cell But now neurons are derived from fibroblasts !
- 46. Marius Wernig et al.demonstrated that fibroblasts convert to functional neurons in vitro by forced expression of three factors:- Ascl1, Brn2, and Myt1l, bypassing the pluripotent stage, thus avoiding the risk of tumor formation. ? •MAP2, NeuN, and synapsin positive, •Produces action Brn2, Myt1l & Ascl1 potentials 12 days mEF iNs Nature. Published online January 27, 2010. doi:10.1038/nature08797
- 47. Instead of using retroviruses, researchers have tried using: Plasmid expression vector- Yamanaka S. et al ADVANTAGE- no evidence of transgene integration, no risk of transformation due to integration. DISADVANTAGE- low efficiency Adenoviral vectors- Konrad H. et al ADVANTAGE- no evidence of transgene integration, no risk of transformation due to integration. DISADVANTAGE- products get diluted out during successive cell divisions in progeny cells; efficiency poor Proteins channelized to cell via poly arginine anchors- Ding S. et al ADVANTAGE- no evidence of transgene integration, no risk of transformation due to integration. DISADVANTAGE- products get diluted out during successive cell divisions in progeny cells; efficiency poor c-MYC was avoided due to its proto-oncogenic nature- Yamanaka S. et al the cells took longer time to become iPSCs; low efficiency
- 48. NATURE METHOD OF THE YEAR : 2009-> iPS Cells
- 49. What are the exact sequence of molecular events that leads to this dramatic reprogramming? Whether additional changes, beyond the expression of the four transcription factors, are involved or not? The process of reprogramming is slow — colonies take up to 20 days to develop into real ES-like cells, and their frequency is quite low. Is this because only a few cells happen to express the right combination or levels of the four factors because of the random integrations of the retroviruses? Or, are there additional events, perhaps associated with retroviral insertion, that are required for full transformation?
- 51. “This will be the long-term solution”- Sir Martin Evans “This is the future of stem cell research. Its hundered times more interesting than SCNT”- Dr. Ian Wilmut “A decade from now, this (hESC) controversy will be just a funny historical footnote.” – Dr. James Thompson. “Embryonic stem cells are not safe, but at the moment, iPS cells are more dangerous.” – Shinya Yamanaka
- 52. Breakthrough of the year-2008; Science 322 ;2008;1766-1767 Catherine Verfaillie; The undoing of differentiation by four defined factors: A big step forward towards generating patient specific pluripotent stem cells; Journal of Hepatology 49 (2008) 871–878 Daley G. Q. et al; Reprogramming of human somatic cells to pluripotency with defined factors; Nature, 451, 2008; 141-148 Dausman A.,Jaenisch R. et al; Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes to Pluripotency; , 2008; Cell 133, 250–264. Dirk D. et al; Induced pluripotency with endogenous and inducible genes; Experimental Cell Research 314 (2008) 3255–3263 Douglas A. et al; Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2; Nature Biotechnology 26, 2008; 1269-1278 Hongyan Z, Shili W et al; Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins; Cell Stem Cell 4, 2009;381-385 Holm Z. and Hans R.; Induction of Pluripotency: From Mouse to Human; Cell 131, 2007; 834-836 James A. T. et al; Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells; Science 318,2007 ; 1916-1919 Keisuke O, Ichisaka T & Shinya Y; Generation of germline-competent induced pluripotent stem cells; Nature 448, 2007; 313-319 Konrad H. et al ; Defining Molecular Cornerstones during Fibroblast to iPS Cell Reprogramming in Mouse; Cell Stem Cell 2, 2008, 230–240 Marius W. et al; Nature. Published online 2010; doi:10.1038/nature08797
- 53. Matthias S.,Maherali N., David T.& Hochedlinger K; Defining Molecular Cornerstones during Fibroblast to iPS Cell Reprogramming in Mouse; March 2008; Cell Stem Cell 2, 230–240, 2008 Method of the Year 2009; Nature Methods.7, 2010, DOI:10.1038/Nmeth.f.294 PAUL S. KNOEPFLER; Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine; Stem Cells 2009;27:1050–1056 Rudolf J. et al; Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic Cells; Cell Stem Cell 2, 151–159,2008; 151-160 Shinya Yamanaka; A Fresh Look at iPS Cells ; Cell 137, 2009 ;13-17 Shinya Yamanaka et al; Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors; Science 322, 2008; 322-328 Varas, Stadtfeld, Aguayo A et al; Stem Cells 2009; 27:300–306 Wernig M., Meissner A. , Cassady J. P. & Jaenisch R.; c-Myc Is Dispensable for Direct Reprogramming of Mouse Fibroblasts ;Cell Stem Cell 2, 2008, 10-14 Xiao-yang Z. et al; iPS cells produce viable mice through tetraploid Complementation; Nature, doi:10.1038/nature08267 Yamanaka S. et al; Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors; Cell 131, 861–872, 2007; 861-873 Zhang X. et al; Myc influences global chromatin structure; The Embo Journal (2006) 25, 2723–2734
Editor's Notes
- #5: Ipsc have made a lot of achievement but it was hard to show all of them so I have selected only the very important onesMouse-> skin cardiomyocyte even sperm produced through this tech !Human-> fibroblast blah blahblah
- #28: WASHINGTON (Reuters) - In a surprise result that can help in the understanding of both aging and cancer, researchers working with an engineered type of stem cell said they reversed the aging process in a rare genetic disease.In this case, they wanted to study a rare, inherited premature aging disorder called dyskeratosis congenita. The blood marrow disorder resembles the better-known aging disease progeria and causes premature graying, warped fingernails and other symptoms as well as a high risk of cancer.It is very rare and normally diagnosed between the ages of 10 and 30. About half of patients have bone marrow failure, which means their bone marrow stops making blood and immune cells properly.But, reporting in Thursday's issue of the journal Nature, they said the process of making the iPS cells appeared to reverse one of the key symptoms of the disease in the cells.In this disease, the cells lose telomerase, an enzyme that helps maintain the telomeres. These are the little caps on the ends of the chromosomes that carry the DNA.A gene called TERC helps restore the telomeres and Daley's team said it may be that tumor cells make use of TERC to become immortal.In making the iPS cells and getting them to grow in the lab, Daley's team discovered they had three times as much TERC as the diseased cells they were made from.Simply turning the skin cells into iPS cells helped restore their damaged telomeres, Daley's team reported. This in theory stops a major component of the aging process as well."We're not saying we've found the fountain of youth, but the process of creating iPS cells recapitulates some of the biology that our species uses to rejuvenate itself in each generation," Daley's colleague Suneet Agarwal said in a statement.